The CDC has documented the worsening ?opioid overdose epidemic? in the U.S. Opioid overdose deaths have increased among males and females, non-Hispanic whites and blacks, and all age groups over age 25.24 Interrelated trends contributing to the epidemic include an increase in prescription opioid overdose deaths spanning more than 15 years and more recent surges in overdose deaths due to illicit opioids (i.e., heroin and fentanyl-related drugs). Overdose deaths involving heroin rose nearly five-fold in the U.S. from 2010 to 2016 while those involving fentanyl and its analogues more than doubled from 2015 to 2016.24,26 Research to improve understanding of the pathophysiology of opioid-induced respiratory depression is thus a public health imperative. Prior genetic research in this area has been limited to candidate gene studies that genotyped a handful of SNPs in samples of very modest size that consisted primarily of European ancestry (EA) individuals. We are proposing to conduct the first GWAS of death due to opioid-induced respiratory depression. Our large EA and African American (AA) sub-samples will each exceed that of the largest prior study by well over an order of magnitude. We will utilize two distinct comparison groups, large previously-genotyped EA and AA samples of opioid dependent individuals and general population members that are well-suited, respectively, to identify effects contingent upon repeated use and those shared with liability of opioid use disorder. Our study design focuses on a definitive outcome measure, death due to opioid-induced respiratory depression, which is expected to provide additional power for our investigation. Many prior studies have examined less clearly demarcated outcomes (clinically observed respiratory depression) in convenience samples (e.g. pediatric surgical patients). This revised proposal is very well-powered to identify common genetic variants in EAs and AAs associated with liability for opioid-induced respiratory depression. We are also proposing to conduct the first examination performed in human opioid overdose decedents of gene expression changes in brain regions involved in opioid-induced respiratory depression including the preBtzinger complex, parabrachial/Klliker-Fuse nuclei (PB/KF), and raphe nucleus. This complementary investigation will provide additional insight into the pathophysiology underlying this process and enable examination of alterations in gene expression associated with common variants implicated in the GWAS. The specific aims are: 1) To obtain DNA from 15,000 accidental opioid overdose decedents (N's EA ~10,000; AA ~5000). 2) To conduct a GWAS of opioid-induced respiratory depression including risk shared across opioids, and drug-specific effects, comparing these individuals to two large previously GWAS-genotyped groups of AA and EA individuals: 1) those with opioid use disorder; and 2) those ascertained in general population samples. 3) To examine differential gene expression in the medullary preBtzinger complex and raphe nucleus, and pontine PB/KF nuclei in those who died from accidental opioid overdose (N=50) compared with separate matched groups who died from other causes: i) those with documented opioid use (N=50); and ii) decedents who had negative toxicology screens (N=50) and to relate differential expression in these regions to GWAS data for Aim 2, providing a biological foundation for novel GWAS discoveries.